Nano-emulsion of 5-aminolevulinic acid

ABSTRACT

The present invention relates to a composition comprising a nano-emulsion that contains 5-aminolevulinic acid as well as a carrier in an aqueous phase. This invention also relates to a pharmaceutical preparation containing this composition. The nano-emulsions of this type can be used in photodynamic therapy as well as in the photodiagnostic detection of proliferatives cells.

This application is a 371 of PCT/EP99/08711 filed Nov. 12, 1999.

The present invention relates to nanoemulsions which contain5-aminolevulinic acid or its derivatives, precursors or metabolites.

Photodynamic therapy is a novel and promising method for treatingvarious premalignant and malignant diseases which are connected to cellproliferation. The principle of photodynamic therapy is based onintroducing what is termed the photosensitizer into the tumor tissue andusing irradiation with light of a suitable wavelength to convert thisphotosensitizer into a cytotoxically active compound which in the enddestroys the cells. The selectivity of this method is based on thesensitizer being concentrated to a greater extent in rapidlyproliferating tumor cells than in normal tissue. Irradiation with lightin a locally restricted manner can then be used to specifically activatethe sensitizer which is present in the tumor cells, thereby destroyingthe cancer cells while to a large extent sparing the healthy tissue.

Until now, an intravenously administered mixture of hematoporphyrinderivatives has in the main been used as the photosensitizer. Despitethe encouraging clinical successes which have been achieved inconnection with a number of different types of cancer, thesehematoporphyrin derivatives nevertheless suffer from a variety ofdisadvantages. In the first place, relatively high concentrations of theactive compound appear in normal tissue due to the low degree of tumorselectivity and the fact that the active compound is only slowlyeliminated from the body. Undesirable photochemical reactions thereforetake place in healthy tissue in connection with the irradiation. In thesecond place, this treatment results in a general sensitivity to lightsuch that the patient is not allowed to expose himself to daylight for aperiod of some four weeks.

In certain cases, it is possible, particularly in connection withdermatological and gynecological applications, to bring about areduction in the high concentration of active compound in normal tissue,and therefore in the undesirable side-effects, by developing topicallyapplicable active compound formulations in place of the known systemicformulations. Attempts are also being made to reduce the sensitivity tolight by using photosensitizer precursors which are photochemicallyinactive and are only converted into a photosensitizer within the targetcell.

5-Aminolevulinic acid is an endogenous substance which is synthesizedfrom glycine and succinyl-CoA. In heme biosynthesis, the extremelyphotoactive protoporphyrin IX is formed from 5-aminolevulinic acid(5-ALA) in several rapidly proceeding reactions steps, and is thenconverted into heme in a slow reaction. If the heme concentration is toohigh, a natural control mechanism inhibits both the endogenous synthesisof 5-aminolevulinic acid and the breakdown of protoporphyrin IX.

This control mechanism is circumvented by exogenously administeringsynthetically prepared 5-aminolevulinic acid, thereby giving rise to anincreased production of protoporphyrin IX. Since the breakdown ofprotoporphyrin IX is still inhibited by the natural control mechanism,this compound becomes concentrated in the cells. When irradiated withlight, protoporphyrin IX is able to enter into a photochemical oxidationreaction and consequently acts as a photosensitizer. When the sensitizermolecule absorbs a quantum of light, it is first of all transferred intoan electronically excited state (singlet state), which is relativelyshort-lived, and either releases its excess energy once again within afew nanoseconds by emitting a fluorescence photon or else passes overinto a relatively long-lived triplet state. Energy from this tripletstate can be transferred to oxygen molecules which are present in thecell. The singlet oxygen which is formed in this connection has acytotoxic effect, in particular on proliferating cells, since it reactswith cell components, for example the cell membrane and themitochondria, or triggers the formation of cell-damaging free radicals.Furthermore, irradiation of the photosensitizer gives rise to acharacteristic fluorescence radiation which can be used for detectionreactions, for example for detecting proliferating cells.

A number of investigations using topically applicable 5-aminolevulinicacid compositions are known from the prior art. While theseinvestigations have the feature in common that the 5-aminolevulinic acidemployed is in the form of an oil-in-water emulsion, differences existwith regard to other parameters, such as period of penetration, periodof treatment, type of light employed and the dose of light applied.

B. Thiele et al. (H+G, Volume 69, No. 3, pages 161-164 (1994)) describeinvestigations which involve using 20% δ-aminolevulinic acid in the formof an oil-in-water emulsion, with a penetration period of from 5 to 6 h,and subsequently irradiating with an argon ion-pumped dye laser(emission maximum 630 nm) giving a cumulative total dose of from 50 to100 J/cm².

Wolf et al. (Journal of the American Academy of Dermatology Vol. 28,pages 17 to 21, 1993) describe investigations which involve using 20%5-aminolevulinic acid in the form of an oil-in-water emulsion, with apenetration period of 4, 6 or 8 h, and irradiating with unfiltered lightor red light, giving a light dose of from 30 J/cm² to 100 J/cm².

Although the investigations disclosed in the prior art clearlydemonstrate the promising potential of photodynamic therapy using5-aminolevulinic acid, oil-in-water emulsions which are so far knownsuffer from a number of disadvantages.

Thus, M. Novo Rodriguez et al. (SPIE, Vol. 2371, pages 204-209) showedthat, in the high concentrations which are required for a clinicalapplication, aminolevulinic acid is unstable in aqueous solutions in theneutral to basic pH range. In the time period of 25 h investigated,satisfactory results are only obtained at a pH of 5.01, and aconcentration of 3% and a pH of 5 are specified as the optimalconditions for aqueous solutions of 5-aminolevulinic acid. However, forclinical use, it will in general also be necessary to providecompositions in a higher concentration range; furthermore, to be usedcommercially, the 5-ALA solutions have to be stable for a period whichis of the order of weeks or months.

V. von Arx et al. (J. Pharm. Pharmacol. 49: 652-656, 1997) describeinvestigations relating to the topical application of 5-aminolevulinicacid in a variety of gels. This publication states that the bestformulation for maintaining the stability of 5-aminolevulinic acid is acombination with Novion AA-1, a polyacrylic acid, at a pH <6.

Another disadvantage of the known oil-in-water emulsions is that thedepth to which the photosensitizer penetrates into the damaged tissue isnot optimal. As a result, the diseased tissue is in many cases onlyaccessible to the photodynamic therapy in its superficial layers eventhough the depth to which the light employed for activating thephotosensitizer penetrates would also enable more deeply lying layers tobe treated.

The object of the present invention was therefore to make available5-aminolevulinic acid-comprising compositions in which the disadvantagesknown from the prior art are at least partially eliminated and which, inparticular, possess adequate stability and exhibit an improved abilityto penetrate into tissue.

This object is achieved by a composition which is characterized in thatit contains a nanoemulsion which comprises a substance selected from5-aminolevulinic acid, or a derivative, a precursor and/or a metabolitethereof, and a carrier in an aqueous phase.

It was observed, surprisingly, that the stability of 5-aminolevulinicacid can be substantially increased when the acid is formulated into ananoemulsion. While the reasons for this are not known, it appears thata microenvironment created by nanosomes has a particularly favorableeffect on the stability of the 5-aminolevulinic acid.

It has furthermore been shown, surprisingly, that very high tissuepenetration depths can be achieved with the nanoemulsions according tothe invention, resulting in more deeply lying diseases, or diseases withhigher layer thicknesses, also becoming accessible to treatment. Thegreater penetration depths were particularly surprising because it hadpreviously been assumed that, due to its small size, 5-aminolevulinicacid would in any case be readily able to penetrate through a damagedepidermis which is present, for example, over inflammations,precancerous stages and tumors.

A third surprising advantage is that, when packed into nanosomes inaccordance with the invention, 5-aminolevulinic acid is evidently takenup very efficiently by the cells. This firstly improves targeting;secondly, it means that the penetration period, i.e. the time betweenapplying the composition and irradiating the diseased tissue with light,can be reduced, with this representing a distinct relief for thepatient.

According to the invention, the nanoemulsion comprises an activesubstance which is selected from 5-aminolevulinic acid or a derivative,a precursor and/or a metabolite thereof. “Derivative” is to beunderstood as being, in particular, salts, complexes and additioncompounds. “Precursor” and “metabolite” are in this connection to beunderstood as being those substances which are converted in a cell intoprotoporphyrin IX. Particular preference is given to the activesubstance being 5-aminolevulinic acid or one of its derivatives. Thecarrier can be any carrier as long as it is able to form thenanoemulsion in an aqueous phase. The carrier preferably comprises anoil phase, i.e. a material which is immiscible with water, for examplelipids, and an emulsifier. Physiologically harmless carrier substancesare expediently used.

The size of the emulsified particles in the nanoemulsion (nanosomes) ison average ≦200 nm, e.g. from 10 to 200 nm. The particle size which isin each case optimal depends on other parameters such as the viscosityof the composition. For example, good results were obtained with a gelhaving a viscosity of 5 mPas at an average particle diameter of about110 nm, and also for a lotion having a viscosity of 1.6 mPas at anaverage particle diameter of about 20 nm.

Suitable carrier systems, which are stable over a long period of time,which do not contain any high concentrations of surfactants andcosurfactants, and which are free from toxic emulsifier complexes, aredisclosed, for example, in U.S. Pat. No. 5,152,923. These nanoemulsionscomprise a glycerophosphatide, such as a lecithin or a cephalin, as theemulsifier and physiologically tolerated lipids, e.g. triglycerides,such as vegetable or animal oils, for example groundnut oil, soybeanoil, etc., as the oil phase. The emulsifier/oil weight ratio is from0.05 to 0.4:1.

Examples of emulsifiers which have already been employed successfully inpractice in 5-aminolevulinic acid nanoemulsions are egg lecithin,soybean lecithin and phosphatidyl choline. An example of an approvedlipid is Miglyol 812.

The proportion of active substance, for example 5-aminolevulinic acid,in the composition essentially depends on the application which isenvisaged. In general, from about 1 to 25% by weight, based on the totalweight of the composition, are present. However, it is also possible touse higher or lower doses. A proportion of from 5 to 15% by weight, inparticular of about 10% by weight, has proved to be suitable forapplications in connection with photodynamic therapy.

The composition can additionally comprise adjuvants and/or additives, inparticular those substances which are customary in cosmetics orpharmacy. Examples of such substances are buffers, stabilizers,additional emulsifiers, thickeners, etc.

In a particularly preferred embodiment, the composition according to theinvention is a gel which, based on the total weight of the composition,comprises from 1 to 25% by weight, preferably from 5 to 15% by weight,of active substance, from 40 to 60% by weight, preferably from 45 to 55%by weight, of carrier and from 0 to 10% by weight, preferably from 1 to5% by weight, of adjuvants, with the remainder being water.

According to another particularly preferred embodiment, the compositionaccording to the invention is a lotion which, based on the total weightof the composition, comprises from 1 to 25% by weight, preferably from 5to 15% by weight, of active substance, from 10 to 30% by weight,preferably from 15 to 25% by weight, of carrier and from 10 to 30% byweight, preferably from 15 to 25% by weight, of adjuvants, with theremainder being water.

As mentioned at the outset, the 5-aminolevulinic acid compositionaccording to the invention exhibits a surprisingly high degree ofstability on storage, with the proportion of active substance in thecomposition having a pH of between 1.5 and 3 preferably being reduced,after one year of storage at room temperature, by not more than 5% and,particularly preferably, by not more than 4%. After one year of storageat 5° C., the proportion of active substance is preferably reduced bynot more than 3% and particularly preferably by not more than 2.5%.

The present invention also relates to the composition according to theinvention which is in the form of a pharmaceutical preparation. In thiscase, the composition is free of constituents which are notpharmaceutically acceptable and preferably free of constituents which,for example, provoke irritation. In addition to the carrier substanceswhich have already been mentioned, the pharmaceutical preparation canalso comprise further adjuvants and/or additives which are acceptableand preferably well tolerated.

The pharmaceutical preparation can be present in a form which issuitable for systemic administration, such as an injectable liquid.However, for dermatological and gynecological applications, the thepreparation is preferably in a form which is suitable for topicaladministration. The preparation possesses properties, e.g. viscosity andrheology, which are favorable for the administration form which is ineach case required in order to ensure that, after the preparation hasbeen administered, the nanosomes loaded with 5-aminolevulinic acidpenetrate to an adequate extent into the target tissue. These viscosityand rheology properties can be adjusted by adding thickeners such aspolyethylene glycol stearyl ethers, polyethylene glycol stearates and/orpolysaccharides such as polysaccharide B-1459, for example.

The present invention also relates to a process for producing thecomposition or the pharmaceutical preparation according to theinvention. In this process, the constituents of the carrier material areinitially introduced in an aqueous phase and the mixture is convertedinto a nanoemulsion by homogenizing thoroughly. It is possible, forexample, to use commercially available high pressure homogenizers forthis purpose. The 5-aminolevulinic acid, and any additives which may bepresent, can be added before and/or after the homogenization. After thenanoemulsion has been prepared, it is then possible to add otheradjuvants and additives whose presence was not desirable during thehomogenization.

Preference is given to excluding air while carrying out the process, forexample by means of applying a vacuum and/or a protective gasatmosphere. In addition, it is preferred to implement the process whileexcluding light. The process is carried out at a temperature at whichthe desired nanoemulsion can be formed and the constituents, inparticular the active substance, is adequately stable. In general, ithas been found that a temperature range of from about 5 to 45° C. issuitable. However, adjuvants and/or additives which are, for example,first of all mixed, and homogenized where appropriate, in a separatemixture, and only after that added to the composition, can be processedat higher temperatures, for example up to about 80° C. For apharmaceutical application, care is taken to ensure that the resultingproduct is sterile, for example by employing sterile starting materialsand maintaining sterile process conditions and/or by inserting asterilization step after the preparation.

An important area of use for the compositions according to the inventionis in the field of photodynamic therapy, with particular preferencebeing given to applying the nanoemulsion topically. The nanoemulsionaccording to the invention can be used in association with all diseaseswhose control comprises inhibiting the proliferation of, or destroying,cells or tissues by photoactivating a sensitizer which is formed from5-aminolevulinic acid. The diseases include, in particular, those whichare associated with an increase in cell proliferation since, in thiscase, the photosensitizer is concentrated to a particularly high degreeby the increased cell metabolism in diseased cells.

The compositions according to the invention are consequently suitablefor treating tumor diseases such as basal cell carcinoma, squamous cellcarcinoma, Bowen's disease, solar keratosis, condylomata acuminata(CIN), epithelial neoplasia of the vulva (VIN), and nodose andsubcutaneous cancer diseases. Psoriasis is an example of a nontumorousdisease.

The treatment is effected, for example, by topically applying ananoemulsion which contains the active substance, e.g. 5-aminolevulinicacid, and then incubating in order to allow an adequate quantity of the5-aminolevulinic acid to penetrate into the tissue which is beingtreated. During the incubation, irradiation of the treated area withlight is preferably avoided, for example by covering it, in order toprevent any undesirable premature activation. After the incubationperiod, which is generally from about 1 to 8 h and usually about 4 h,has expired, the tissue is irradiated with an adequate dose of radiationusing a light source. Suitable light sources include lamps which emitwhite light and also monochromatic light sources, such as a laser, inparticular an argon dye laser which emits at about 630 nm. The radiationdoses are normally in a range of from about 20 J/cm² to several hundredJ/cm² per application.

Another area for using the nanoemulsions according to the inventionrelates to detecting the presence of proliferating cells in a sample,for example a tissue sample. The detection is based on selectivelyconcentrating a photosensitizer, which is produced by metabolism of theactive substance, in proliferating cells as compared with normal cells.Preference is given to the active substance being 5-aminolevulinic acidand the photosensitizer being protoporphyrin IX. The extent to which thephotosensitizes has been concentrated can be determined by means ofphotodiagnostic methods, for example by irradiating with light having awavelength of 405 nm and measuring the fluorescence radiation generatedby the photosensitizer. The nanoemulsions according to the invention areparticularly suitable for being used in tumor diagnosis.

The invention furthermore relates to the use of the nanoemulsionaccording to the invention for producing a drug for photodynamictherapy.

Finally, the invention relates to a kit which comprises a nanoemulsionaccording to the invention, which is suitable for being appliedtopically, and one or more auxiliary substances. Examples of theseauxiliary substances are a covering material, such as a plastic filmwhich is applied to the site being treated, after the nanoemulsion hasbeen applied, in order to prevent premature activation by light, andmeans for attaching the covering material or else means for applying thenanoemulsion to the site being treated.

The following examples are intended to clarify the invention.

EXAMPLES 1. Preparing a 10% 5-Aminolevulinic Acid Lotion

A nanocolloid carrier system was prepared, in a phosphate buffer, fromegg lecithin (83% phosphatidylcholine), Miglyol 812 (triglyceride) andPolysorbatum 80 using the method described in U.S. Pat. No. 5,152,923.The analytical data for the carrier system were as given in table 1.

TABLE 1 Aqueous phase 20 mM phosphate buffer, pH 6.0 optical propertiesyellow, highly iridescent liquid pH at room temperature 6.0 viscosity(20° C.) 1.6 mPas size of the nanoparticles ≦10-200 nm average diameter19.4 nm content of egg lecithin 17.5 mg/ml content of Polysorbatum 80≦3% (w/w) content of Miglyol 812 34-38 mg/ml aerobic mesophilic <1CFU/ml organisms in 50 ml

The components used for preparing a 5-ALA-nanocolloid lotion, and theirrelative proportions, are given in table 2.

TABLE 2 Quantity Name (% by weight) Phase 1 Cetyl alcohol 5.00% Stearylalcohol 1.00% Glycerol monostearate 2.00% SNOWWHITE vaseline grease2.00% Pharm. perliquidum paraffin 2.00% Cosm. isopropyl myristate 4.00%Cremophor A 25 1.00% Cremophor S9 1.50% Cremophor EL 00647 1.88% Phase 2Water 48.87% Sorbitol, 70% 0.25% Phenoxyethanol 0.50% Phase 3Nanocolloid (table 1) 20.00% 5-Aminolevulinic acid hydrochloride 10.00%

All the procedural steps were carried out while excluding light andatmospheric oxygen. Phase 1 was prepared by melting together, at 80° C.,the components shown in table 2, in the given quantity ratios, and thenmixing.

Phase 2 was prepared in a separate receptacle. For this, the water wasintroduced initially and the remaining components shown in table 2 wereadded while stirring. After it had been adequately mixed, phase 2 washeated to 80° C. and admixed with phase 1 in vacuo.

After it had been cooled down to 75° C., the mixture was homogenized for2 min in a homogenizer. The resulting mixture was cooled down to 60° C.and homogenized once again for 2 min.

Phase 3 was prepared in a separate receptacle in vacuo and whileexcluding light. For this, the nanocolloid carrier system, as describedabove, was introduced initially and the 5-aminolevulinic acidhydrochloride was dissolved in it at from 25 to 30° C. Phase 3 was thenadded, at 40° C. and in vacuo, to the mixture of phases 1 and 2. Afterthat, the composition was gassed with protective gas and homogenized forfrom 2 to 3 min in a homogenizer. It was then left to cool down to roomtemperature while being stirred.

In order to determine the long-term stability of the active compound, aportion of the lotion was subjected to a storage test. Following oneyear of storage at 5° C., the content of 5-ALA was 97.92% of theoriginal content, while a value of 96.50% was obtained at roomtemperature over the same period of time.

2. Preparing a 10% 5-Aminolevulinic Acid Gel

A nanocolloid carrier system was prepared, in a K/Na phosphate buffer,from egg lecithin and Miglyol 812 (triglyceride) using the methoddescribed in U.S. Pat. No. 5,152,923. The analytical data were as givenin table 3.

TABLE 3 20 mM phosphate buffer, Aqueous phase pH 6.0 optical propertiesmilky liquid pH at room temperature 6.0 viscosity at 20° C. 1.5 mPassize of the nanoparticles ≦10-200 nm average diameter 110.6 nm standarddeviation 32.1% content of total lipid 105.4 mg/g content of lecithin27.0 mg/g content of Miglyol 812 78.4 mg/g aerobic mesophilic organisms<1 CFU/ml in 100 ml

The components used for preparing a 5-aminolevulinic acid nanocolloidgel, and their relative proportions, are given in table 4.

TABLE 4 Quantity Name (% by weight) Phase 1 Water 38.30% Keltrol 1.70%Phase 2 Nanocolloid (table 3) 50.00% Aminolevulinic acid 10.00%hydrochloride

In order to prepare phase 1, the water was introduced initially andheated to from 60 to 70° C., after which the Keltrol was dispersed in itin vacuo. The mixture was homogenized for 4 min at step 1, after whichit was left to cool down to 30° C. while being stirred at step 1.

In order to prepare phase 2, the nanocarrier system was initiallyintroduced in a sealed vessel, in vacuo and at room temperature, and the5-aminolevulinic acid hydrochloride was completely dissolved in it overa period of from 2 to 3 h while the mixture was being stirred.

After that, phase 2 was mixed with phase 1 in vacuo and the whole wasthen gassed with nitrogen. The resulting composition was mixed tohomogeneity while being stirred for 2 h at a temperature of at most 30°C.

In order to determine the long-term stability of the active compound, aportion of the gel was subjected to a storage test. After one year ofstorage at 5° C., the content of 5-ALA was 99.17% of the originalcontent, while a value of 98.94% was obtained at room temperature overthe same period of time.

3. Photodynamic Therapy Using the Nanocolloid Lotion Described inExample 1

The effect of the nanolotion according to the invention was investigatedin a clinical study of 55 basal cell carcinomas in a group of patientsconsisting of 19 individuals.

Before the nanocolloid lotion was applied, the entire skin area to betreated was cleaned with an alcoholic solution. In each case 0.15 g ofnanocolloid solution was applied per cm² of the skin area to be treated,resulting in a thin, visible film of lotion. After the lotion had beenapplied, the entire area was covered with a light-impermeable coveringmaterial in order to prevent the lotion smearing and to prevent anyundesirable photodynamic reactions brought about by the light in theroom. After a reaction time of 6 h, the covering was removed and thepresence of protoporphyrin IX, and the extent of the tumor, wereassessed on the basis of the characteristically red fluorescence ofporphyrins when irradiated with ultraviolet light.

The irradiation was performed using unfiltered light from a 250 Whalogen lamp having a spectral distribution over the entire visiblerange with a maximum of about 800 nm. All the lesions were irradiated ata distance of 10 cm, thereby enabling regions having a diameter of up to10 cm to be irradiated. The time of irradiation was 20 min, and theintensity was 200 mW/cm², corresponding to a total light dose of 240J/cm².

The group of patients comprised 19 individuals who had one or moresuperficial basal cell carcinomas without metastases. In all, 55 basalcell carcinomas were treated by photodynamic therapy. None of thepatients included in this study had previously been treated either witha conventional method or with photodynamic therapy. The patients wereaged from 32 to 93, with the average age being 65. 14 (73.7%) patientswere male and 5 (25.3%) were female. With one exception, the skin tumorswhich were treated in this study were superficial lesions. The averagediameter of the lesions was 13.2 mm, with the size varying between 4.0and 45 mm. The number of tumors treated in the different body regionswas 11 (20%) in the head and neck region, 37 (67%) in the trunk region,3 (5%) on the upper limbs and 4 (7%) on the lower limbs. Before thetreatment was initiated, biopsies were routinely removed for confirmingthe diagnosis. The success of the therapy was assessed by visualinspection and palpation and, in the case of 26 (47%) of the tumors, byhistopathological investigations as well. The absence of a clinicallydetectable tumor at the treatment site at the time of the postirradiation examination was defined as being a complete tumor response.A perceptible reduction in the size of the tumor was defined as being apartial tumor response.

The response rates which were achieved in this study are summarized intable 5. It was found that 47 (85%) of the 55 basal cell carcinomastreated in the 19 patients regressed completely after one singletreatment, as was confirmed clinically by subsequent investigationperformed at least 6 months after the treatment. The remaining 8 (15%)basal cell carcinomas were found to have regressed partially, i.e. therewas a perceptible reduction in the size of the tumors.

Table 6 shows how the results of the photodynamic therapy varied withthe location of the basal cell carcinomas. The best results wereobtained for the seven limb lesions, all of which regressed completely.Of the 37 trunk lesions, 32 tumors regressed completely, while 8 (76%)of the 11 tumors in the head and neck region also regressed completely.

TABLE 5 Visual assessment Biopsy Number of patients 19 13 Basal cellcarcinomas 55 26 Complete regression 47 (85%) 21 (81%) Partialregression  8 (15%)  5 (19%) No reaction — —

TABLE 6 Head and neck Trunk Limbs Number of patients 6 12 4 Basal cellcarcinomas 11 37 7 Complete regression 8 (73%) 32 (86%) 7 (100%) Partialregression 3 (27%)  5 (14%) — No reaction — — —

4. Photodynamic Therapy Using the Nanocolloid Gel Described in Example 2

The effect of the nanoemulsion according to the invention in the form agel was investigated in a clinical study of the photodynamic therapy ofcondylomata acuminata and intraepithelial neoplasia of the vulva (VIN)as performed on 47 lesions in a group of patients consisting of 16individuals aged from 18 to 45 (average age, 32.7).

The gel was applied as described for the lotion in example 3 except thatthe incubation time allowed for diffusing the 5-aminolevulinic acid wasonly 90 min. Irradiation was carried out using an argon dye laser(Coherent Innova, Model 310, Palo Alto, Calif.) with monochromatic light(630 nm) and with light doses of between 50 J/cm² and 125 J/cm².

After one single treatment, nine of the 16 patients showed completeregression, while the other seven showed partial regression, during apost-irradiation examination period of between 1 and 12 months. Thetreatment was well tolerated by the majority of the group of patients.The patients were free to interrupt the irradiation if the pain becameexcessive. The number of interruptions which occurred prior to thecomplete radiation time being reached was recorded. Only three of thepatients had interrupted the irradiation more than five times, whilefive of the patients interrupted the irradiation once or twice; theremaining patients did not require any interruption.

What is claimed is:
 1. A composition comprising a nanoemulsion whichcomprises an active substance which can be converted into protoporphyrinIX, the active substance being selected from the group consisting of atleast one of 5-aminolevulinic acid, a salt compound thereof, a complexcompound thereof, an addition compound thereof, precursors thereof, andmetabolites thereof, and (b) a carrier in an aqueous phase, with thecarrier being formed from at least one lipid and from at least oneemulsifier comprising soybean lecithin.
 2. The composition of claim 1,wherein the average size of the emulsified particles is from 10 to 200nm.
 3. The composition of claim 1 wherein the active substance ispresent in a proportion of from 1 to 25% by weight based on the totalweight of the composition.
 4. The composition of claim 1 wherein thecomposition additionally comprises adjuvants and/or additives which arecustomary in cosmetics or pharmacy.
 5. The composition of claim 4wherein the composition is present in the form of a gel and, based onthe total weight of the composition, comprises from 5 to 15% of activesubstance, from 45 to 55% of carrier and from 1 to 5% of adjuvants, withthe remainder being water.
 6. The composition of claim 4 wherein thecomposition is present in the form of a lotion and, based on the totalweight of the composition, comprises from 5 to 15% of active substance,from 15 to 25% of carrier and from 15 to 25% of adjuvants, with theremainder being water.
 7. The composition of claim 1 wherein the contentof active substance is reduced by not more than 5% after one year ofstorage at room temperature.
 8. The composition of claim 1 wherein thecomposition is in the form of a pharmaceutical preparation.
 9. Thecomposition of claim 8 wherein the composition can be applied topically.10. A kit which comprises a topically applicable composition as claimedin claim 9 and at least one component selected from the group consistingof: (a) an essentially light-impermeable sheet-like material, (b) meansfor attaching the sheet-like material to a site of application, and (c)means for applying the composition to a site of application.
 11. Aprocess for preparing a composition as claimed in claim 1 wherein amixture comprising a carrier and an aqueous phase is prepared andconverted into a nanoemulsion, with the active substance being addedbefore and/or after the conversion into the nanoemulsion, and, afterthat, adjuvants and/or additives are admixed where appropriate.
 12. Theprocess of claim 11, process is carried out while excluding oxygenand/or light.
 13. The process of claim 11 wherein the process is carriedout at a temperature of from 5 to 45° C.
 14. A method of photodynamictherapy comprising topically applying a nanoemulsion that contains thecomposition of claim 1; then incubating in order to allow thecomposition of claim 1 to penetrate into tissue that is being treated;then irradiating the tissue with radiation.
 15. The method of claim 14wherein the tissue being treated has a disease associated with cellproliferation.
 16. The method of claim 15, wherein the disease is atumor disease.
 17. The method of claim 16, wherein the disease is abasal cell carcinoma, a squamous cell carcinoma, Bowen's disease, solarkeratosis, condylomata acuminata (CIN), intraepithelial neoplasia of thevulva (VIN), or a nodose or subcutaneous cancer disease.
 18. The methodof claim 15, wherein the disease is psoriasis.
 19. A process forphotodynamic therapy, wherein a composition as claimed in claim 1 isadministered in an effective quantity to a diseased organism, incubationis performed for a period which is suitable for ensuring that anadequate quantity of the active substance is present in the tissue beingtreated, and thereafter the tissue is irradiated with light.
 20. Amethod for detecting proliferating cells comprising selectivelyconcentrating a photosensitizer that is produced by metabolism of thecomposition of claim 1 in proliferating cells as compared with normalcells; and then determining an extent to which the photosensitizer hasbeen concentrated.
 21. The method of claim 20 wherein proliferatingcells are those of tumor diseases.